Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb, which has been well-characterized through studies in classical genetics and molecular biology (Horwitz, M. S., "Adenoviridae and Their Replication," in Virology, 2nd edition, Fields et al., eds., Raven Press, New York, 1990). The viral genes are classified into early (known as E1-E4) and late (known as L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins. The demarcation between these events is viral DNA replication.
Recombinant adenoviruses have several advantages for use as gene transfer vectors, including tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (Berkner, K. L., Curr. Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Ther. 1:51-64, 1994).
The cloning capacity of an adenoviral vector is proportional to the size of the adenovirus genome present in the vector. For example, a cloning capacity of about 8 kb can be created from the deletion of certain regions of the virus genome dispensable for virus growth, e.g., E3, and the deletion of a genomic region such as E1 whose function may be restored in trans from 293 cells (Graham, F. L., J. Gen. Virol. 36:59-72, 1977) or A549 cells (Imler et al., Gene Ther. 3:75-84, 1996). Such E1-deleted vectors are rendered replication-defective. The upper limit of vector DNA capacity is about 105%-108% of the length of the wild-type genome. Further adenovirus genomic modifications are possible in vector design using cell lines which supply other viral gene products in trans, e.g., complementation of E2a (Zhou et al., J. Virol. 70:7030-7038, 1996), complementation of E4 (Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995; Wang et al., Gene Ther. 2:775-783, 1995), or complementation of protein IX (Caravokyri et al., J. Virol. 69:6627-6633, 1995; Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995). Maximum carrying capacity can be achieved using adenoviral vectors deleted for all viral coding sequences (Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22, 1996).
Transgenes that have been expressed to date by adenoviral vectors include p53 (Wills et al., Hum. Gene Ther. 5:1079-188, 1994); dystrophin (Vincent et al., Nature Genetics 5:130-134, 1993; erythropoietin (Descamps et al., Hum. Gene Ther. 5:979-985, 1994; omithine transcarbamylase (Stratford-Perricaudet et al., Hum. Gene Ther. 1:241-256, 1990; We et al., J. Biol. Chem. 271;3639-3646, 1996;); adenosine deaminase (Mitani et al., Hum. Gene Ther. 5:941-948, 1994); interleukin-2 (Haddada et al., Hum. Genie Ther. 4:703-711, 1993); and .alpha.1-antitrypsin (Jaffe et al., Nature Genetics 1:372-378, 1992); thrombopoictin (Ohwada et al., Blood 88:778-784, 1996); and cytosine deaminase (Ohwada et al., Hum. Gene Ther. 7:1567-1576, 1996).
The tropism of adenoviruses for cells of the respiratory tract has particular relevance to the use of adenovirus in Gene Ther. for cystic fibrosis (CF), which is the most common autosomal recessive disease in Caucasians. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that disturb the cAMP-regulated C1.sup.- channel in airway epithelia result in pulmonary dysfunction (Zabner et al., Nature Genetics 6:75-83, 1994). Adenoviral vectors engineered to carry the CFTR gene have been developed (Rich et al., Hum. Gene Ther. 4:461-476, 1993) and studies have shown the ability of these vectors to deliver CFTR to nasal epithelia of CF patients (Zabner et al., Cell 75:207-216, 1993), the airway epithelia of cotton rats and primates (Zabner et al., Nature Genetics 6:75-83, 1994), and the respiratory epithelium of CF patients (Crystal et al., Nature Genetics 8:42-51, 1994). Recent studies have shown that administering an adenoviral vector containing a DNA sequence encoding CFTR to airway epithelial cells of CF patients can restore a functioning chloride ion channel in the treated epithelial cells (Zabner et al., J. Clin. Invest. 97:1504-1511, 1996).
The use of adenoviral vectors in gene transfer studies to date indicates that persistence of transgene expression is often transient. At least some of the limitation is due to the generation of a host immune response to the viral proteins which are expressed antigenically even from a replication-defective vector, triggering a pathological inflammatory response which may destroy or adversely affect the adenovirus-infected cells (Yang et al., J. Virol. 69:2004-2015, 1995; Yang et al., Proc. Natl. Acad. Sci. USA 91:4407-4411, 1994; Zsengeller et al., Hum Gene Ther. 6:457-467, 1995; Worgall et al., Hum. Gene Ther. 8:37-44, 1997; Kaplan et al., Hum. Gene Ther. 8:45-56, 1997). Immunologic reactions by the host to adenovirus infection include, inter alia, the generation of cytotoxic T-lymphocytes (CTL) which lyse infected cells displaying a viral antigen, cytolysis of virus-infected cells by tumor necrosis factor (TNF), synthesis of interferons, induction of apoptosis, production of antibodies, and other immunologic mechanisms (Smith, G. L., Trends Microbiol. 2:81-88, 1994). Because adenovirus does not integrate into the cell genome, host immune responses that destroy virions or infected cells have the potential to limit adenovirus-based gene delivery. An adverse immune response poses a serious obstacle for high dose administration of an adenoviral vector or for repeated administration (Crystal, R., Science 270:404-410, 1995).
In order to circumvent the host immune response which limits the persistence of transgene expression, various strategies have been employed, generally involving either the modulation of the immune response itself or the engineering of a vector that decreases the immune response.
The administration of immunosuppressive agents together with an adenoviral vector has been shown to prolong transgenc persistence (Fang et al., Hum. Gene Ther. 6:1039-1044, 1995; Kay et al., Nature Genetics 11:191-197, 1995; Zsellenger et al., Hum. Gene Ther. 6:457-467, 1995).
The administration of adenoviral vectors with alternating serotypes has shown some circumvention of the host immune response (Mack et al., Hum. Gene Ther. 8:99-109, 1997). Animal model studies have shown that persistence of transgene expression can vary among different mouse strains (Barr et al., Gene Ther. 2:151-155, 1995).
The lack of persistence in the expression of adenoviral vector-delivered transgenes may be due to limitations imposed by the choice of promoter or transgene contained in the transcription unit (Guo et al., Gene Ther. 3:802-801, 1996; Tripathy et al., Nature Med. 2:545-550, 1996).
Modifications to the adenoviius genomic sequences contained in the recombinant vector have been attempted in order to decrease the host immune response (Yang et al., Nature Genetics 7:362-369, 1994; Lieber et al., J. Virol. 70:8944-8960, 1996; Gorziglia et al., J. Virol. 70:4173-4178; Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22, 1996).
In addition to deletions in the adenovirus E1 region, first-generation adenoviral vectors often contain modifications to the E3 region in order to increase the packaging capacity of the vectors and to reduce viral gene expression (Yang et al., J. Virol. 69:2004-2015, 1995; Zsengeller et al., Hum. Gene Ther. 6:457-467, 1995; Brody et al., Hum. Gene Ther. 5:821-836, 1994). However, the adenovirus E3 regions contains certain proteins which modulate the host's antiviral immune response. The E3 transcription unit encodes the 12.5K, 6.7K, gp19K, 11.6K, 10.4K, 14.5K and 14.7K proteins (Wold et al., Trends Microbiol. 2:437-443, 1994). The E3 14.7K, 14.5K, and 10.4K proteins are able to protect infected cells from TNF-induced cytolysis. The adenovirus E3 gp19K protein can complex with MHC Class I antigens and retain them in the endoplasmic reticulum, which prevents cell surface presentation and killing of infected cells by cytotoxic T-lymphocytes (CTLs) (Wold et al., Trends Microbiol. 437-443, 1994), suggesting that its presence in a recombinant adenoviral vector may be beneficial. The E3 11.6K gene (adenovirus death protein) is required for cell lysis and the release of adenovirus from infected cells (Tollefson et al., J. Virol. 70:2296-2306, 1996; Tollefson et al., Virology 220:152-162, 1996).
Earlier designs of adenoviral vectors in which the E3 region was modified have shown only transient expression of a transgene in the lungs of test animals (Yang et al., J. Virol. 69:2004-2015; Zsengeller et al., Hum Gene Ther. 6:457-467, 1995).
Modifications to the adenovirus E4 region have been introduced into adenoviral vectors in order to reduce viral gene expression and to further increase carrying capacity (Armentano et al., Hum. Gene Ther. 6:1343-1353, 1995). However, experiments in which adenoviral vectors were introduced into nude mice demonstrated that the context of the adenovirus E4 genomic region was a determinant in the persistence of expression, especially when the CMV promoter was used to control expression of the transgene (Kaplan et al., Hum. Gene Ther. 8:45-56, 1997; Armentano et al., J. Virol. 71:2408-2416, 1997).
The current state of adenoviral vector-based gene delivery requires the development of novel adenoviral vectors which demonstrate a capability for persistence and sustained expression of a transgene.